Inhalation anthrax has emerged as a significant and continuing biowarfare and bioterrorism threat. Though vaccine strategies have substantially controlled this disease in animal populations, the current vaccine licensed for human use has several shortcomings, including significant adverse reactions and low immunogenicity resulting in the need for multiple immunizations. The biology of anthrax infection and specifically the actions of its toxins have been elegantly elucidated at the molecular and cellular level. This presents a unique opportunity to rationally design and develop an improved vaccine that can counter current anthrax, as well as modified forms that may be encountered in the future. In this project, we will use the substantial body of knowledge and insight available relating to anthrax toxin to develop molecularly targeted vaccines to prevent the catastrophic effects of toxin following infection. Based on careful analysis of the crystal structure, sequence, and functional studies of all components of the anthrax toxins, we will develop soluble protein vaccines that direct antibody responses towards the critical protein segments involved in toxin activation, assembly, and adherence to cells. We will test these vaccine constructs for their ability to elicit strong antibody responses with the ability to neutralize toxin activity in in vitro assays. Based on analysis of responses to the soluble protein constructs, selected constructs will be moved into the adeno-associated virus vaccine platform for development of an expression vaccine. Traditionally such vaccines have exhibited higher immunogenicity, and AAV in particular has yielded very durable responses. The AAV-based vaccines will be tested for immunogenicity and their ability to elicit toxin-neutralizing antibodies, as well as for use in concert with soluble protein immunogens. Finally, the most promising constructs of both soluble protein and expressed forms will be tested in anthrax spore inhalation challenge studies in rabbits. Such experiments have been extensively validated against primate experiments previously and also appear to correlate most closely with human anthrax disease and protection. Through this approach, we will develop promising candidate anthrax vaccines that can protect against currently existing strains of anthrax, as well as new and enhanced threats based on engineered forms of anthrax.